mTOR is a protein kinase that signals in pathways involved in the control of cell growth and proliferation. mTOR function is stimulated by both insulin/growth factors and certain amino acids; however, just how the essential kinase activity is controlled in cells is a mystery. Inappropriate activation of mTOR leads to insulin resistance, a contributing factor in the pathogenesis of type 2 diabetes. mTOR inhibitors are used as immunosuppressants, and trials are underway to evaluate their efficacy in treating cancer. Clearly, there is a need to understand better the control and function of mTOR. Two mTOR signaling complexes were recently discovered. mTORC1 contains mTOR, mLST8 (homologous to G protein beta subunits), and raptor, which binds substrates phosphorylated by mTOR. Rapamycin inhibits mTORC1, which probably mediates most of the functions now attributed to mTOR. mTORC2 is not inhibited by rapamycin, and the downstream targets of mTORC2 are largely unknown. mTORC2 contains mTOR, mLST8, and pianissimo, a protein originally implicated in the control of adenylate cyclase. We have searched for new mTOR interacting proteins by yeast two hybrid screening and by performing mass spectrometric (MS) analyses of proteins that immunoprecipitate with mTOR. In investigating an interaction involving eIF3, a key translation initiation factor, we discovered a dramatic rapamycin-sensitive action of insulin to stimulate the association of eIF3 and eIF4G. Aim 1 is to investigate this novel and potentially important effect of insulin. Aim 2 is to investigate the function and control of mTORC1 and mTORC2. We will test the hypothesis that insulin increases mTORC1 activity by increasing substrate binding to raptor. Based on preliminary findings, we also propose to test the hypothesis that insulin promotes formation of mTORC2, to identify the phosphorylation sites in pianissimo, and to investigate connections between mTORC2 and cAMP production. Aim 3 is to identify new upstream effectors and downstream targets of mTORC1 and mTORC2. We will investigate novel candidate mTOR-interacting proteins identified by MS. Finally, to understand better the mechanisms involved in mTORC2, we propose to identify pianissimo-interacting proteins.